Biochemical and biophysical characterization of recombinant rat apolipoprotein E: similarities to human apolipoprotein E3.
Biochemical and biophysical characterization of recombinant rat apolipoprotein E: similarities to human apolipoprotein E3.
复制标题
重组大鼠载脂蛋白 E 的生化和生物物理特征：与人载脂蛋白 E3 的相似性。
DOI：
10.1016/j.abb.2012.10.007
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发表时间：
2013
影响因子：
3.9
通讯作者：
Narayanaswami,Vasanthy
中科院分区：
文献类型：
--
作者：
Tran,TuyenN;Kim,SeaH;Gallo,Carlos;Amaya,Max;Kyees,Jessica;Narayanaswami,Vasanthy
载脂蛋白E（apoE）是一种抗动脉粥样硬化蛋白，凭借其作为低密度脂蛋白受体（LDLr）蛋白家族配体的能力，在维持血浆胆固醇和甘油三酯稳态方面发挥关键作用。在这项研究中，我们的特点是重组大鼠载脂蛋白E的生物化学和生物物理特性，与人载脂蛋白E3。大鼠apoE在大肠杆菌中使用密码子优化系统过表达，并通过亲和层析纯化。大鼠apoE的SDS-PAGE和RP-HPLC证实了其纯度，而免疫印迹验证了apoE 3与LDL r结合区的同一性和交叉反应性。用圆二色光谱法测得α-螺旋含量约为45%。该蛋白在无脂质状态下主要以四聚体形式存在。化学变性的研究表明，展开模式是双相的中间点对应于0.8和2.2M盐酸胍，这表明存在两个域的变性。大鼠apoE将DMPC囊泡转化为更小的DMPC/apoE复合物，一级速率常数为0.12 min − 1。它具有结合LDLr和肝素的能力。我们的研究表明，虽然它的序列类似于apoE3的亚型apoE4，但大鼠apoE显示了apoE3的生物物理行为。
Apolipoprotein E (apoE) is an anti-atherogenic protein that plays a critical role in maintaining plasma cholesterol and triglyceride homeostasis by virtue of its ability to act as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. In this study, we characterized the biochemical and biophysical features of recombinant rat apoE, in comparison with those of human apoE3. Rat apoE was overexpressed in Escherichia coli using a codon optimized system and purified by affinity chromatography. SDS–PAGE and RP-HPLC of rat apoE confirmed the purity, while immunoblot verified the identity and cross-reactivity with the LDLr-binding region of apoE3. The α-helical content was calculated to be ∼45% by circular dichroism spectroscopy. The protein exists in a predominantly tetrameric form in lipid-free state. Chemical denaturation studies reveal that the unfolding pattern is biphasic with mid points of denaturation corresponding to 0.8 and 2.2M guanidine hydrochloride, suggesting the presence of two domains. Rat apoE converts DMPC vesicles to smaller DMPC/apoE complexes with a first order rate constant of 0.12min−1. It has the ability to bind the LDLr and to heparin. Our studies Findicate that although its sequence resembles apoE4, an isoform of apoE3, rat apoE displays the biophysical behavior of apoE3.